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Veterinary Dermatology 2005, 16, 261268

Humoral measurement of type-1 hypersensitivity reactions to a


commercial Malassezia allergen

Blackwell Publishing, Ltd.

K. FARVER*, D. O. MORRIS*, F. SHOFER* and B. ESCH


*Department of Clinical Studies, University of Pennsylvania, School of Veterinary Medicine, Philadelphia, PA,
USA, Greer Laboratories, Lenoir, NC, USA
(Received 12 January 2005; accepted 17 May 2005)

Abstract Malassezia pachydermatis is considered to be a contributing factor to canine atopic dermatitis (AD).
The purpose of this study was to investigate the humoral response to a commercially produced M. pachydermatis
extract. Fifteen atopic dogs with Malassezia overgrowth on the skin (MD), 16 atopic dogs without MD, three
atopic dogs with overgrowth of Malassezia in the ears only (MO), and 12 normal dogs were intradermally
tested with M. pachydermatis extract at 50, 100, 250, 500, 1000, 2000 and 4000 PNU mL1. All dogs were
evaluated cytologically by cutaneous tape strip and bilateral ear exudate sampling to determine presence of MD
or MO. Each had serum evaluated for anti-Malassezia IgE using three Malassezia extracts with an ELISA assay.
The irritant threshold concentration at which healthy nonatopic dogs ceased to react was 1000 PNU mL1. There
was a significant difference in intradermal test reactivity between the atopic groups. At this dilution, 93% (14/
15) of the atopic MD group, 31% (5/16) of the atopic group without MD or MO, and 100% (3/3) of the atopic
MO only group reacted. There were no significant differences in the serum IgE levels as measured by the Greer
ELISA assay, between any groups using any of the three extracts. These results support that Greers
M. pachydermatis extract is useful for intradermal testing of dogs with an allergic phenotype, and that atopics
with MD are more likely to have a type-1 Malassezia hypersensitivity than those without. The ELISA assay may
require further development in order to be useful for the diagnosis of Malassezia hypersensitivity.

IN TRO D U CT ION
Malassezia pachydermatis is a nonlipid-dependent
lipophilic yeast which may be isolated from normal
canine skin in low numbers. However, when there are
changes in the microenvironment and natural defence
mechanisms of the epidermis, increased colonization
by M. pachydermatis may occur.13 The number of
organisms that may be demonstrated on the skin of
healthy dogs during cytological analysis is variable
among individual dogs and depends on sampling site
and breed. Normal basset hounds have been shown to
have higher carriage of Malassezia than other dogs.4 In
a previous report, it was demonstrated that < 3 yeast/
1.6 cm2 body surface area is normal in 70% of normal
dogs and < 10 yeast/1.6 cm2 is normal in 95% of normal
dogs5 (Fig. 1). When yeast numbers beyond this figure
are identified on atopic dogs with active dermatitis,
there is cytological and clinical evidence of Malassezia
dermatitis. These dogs predictably respond to antifungal therapy. Cases of Malassezia-associated dermatitis
typically present with marked inflammation and pruritus, and previous studies have demonstrated that atopic
dogs with Malassezia overgrowth can mount cell-mediated
This study was presented at the annual North American Veterinary
Dermatology Forum in Sarasota, FL, USA, April 2005.
Correspondence: Daniel O. Morris, School of Veterinary Medicine,
University of Pennsylvania, 3900 Delancey Street, Philadelphia, PA
19104, USA. E-mail: domorris@vet.upenn.edu
2005 European Society of Veterinary Dermatology

and humoral (immediate; type I) hypersensitivity


reactions to crude extracts of M. pachydermatis.610
Peripheral blood mononuclear cells isolated from atopic
dogs with Malassezia overgrowth exhibited significantly
increased blastogenic responses to M. pachydermatis
extracts compared to those isolated from normal dogs.7
Evidence for immediate hypersensitivity includes increased
intradermal test (IDT) reactivity to crude extracts of
M. pachydermatis, increased serum concentrations of
anti-Malassezia IgE as determined by the use of ELISA,
and the successful passive transfer of cutaneous anaphylaxis via atopic canine serum (which harboured high
titres of anti-Malassezia IgE) to normal dogs.6,8,9 Demonstration of passive transfer by the PrausnitzKustner
test confirms the functionality of canine anti-Malassezia
IgE.9 In addition, western immunoblotting of sera from
dogs with Malassezia overgrowth has identified several
candidate allergens produced by M. pachydermatis that
are recognized by atopic dogs.10
Malassezia-associated dermatitis and otitis are
common clinical problems and are often exacerbated in
conjunction with atopic flares.13 Clinical identification of
atopic dogs with type I hypersensitivity reactions to a
commercially available and standardized M. pachydermatis
extract would be beneficial, as it could assist in choosing dogs that might be candidates for modification of
the dysregulated immune response by allergen-specific
immunotherapy. Both IDT and ELISA techniques are
used in clinical practice. The clinical identification of
hypersensitivity requires that a commercial allergenic
261

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K Farver et al.

Figure 1. Graph of Malassezia yeast population density on normal


canine skin, showing the cumulative percentage of normal dogs
exhibiting less than X number of yeast on direct impression smear,
as described by Kennis et al.5 Yeast counts were limited to 1.6 cm2
area of a glass slide, with microscopic examination performed at 100
under oil immersion. Impressions were taken from two adjacent interdigital webs of a forepaw, the dorsal aspect of a forelimb, one axillary
fossa, the ventrum and dorsum of the neck, the chin, perioral region,
inguinal region, and umbilical region. The slide with the greatest
number of yeast was chosen from each dog for graphic analysis.
Figure reprinted with permission from an article by Morris et al.8

extract be used for IDT at the optimal concentration for


detecting true sensitization. The optimal concentration
of an allergen is the highest concentration that fails
to produce a reaction in 90% of normal individuals
(the irritant threshold), but that is capable of identifying a significant number of clinically allergic individuals (the allergy detection threshold).11,12 Testing with
an allergen at a concentration above the threshold may
result in a nonspecific false positive (irritant) reaction.
The purposes of this study were to evaluate the
canine humoral response to a new USDA-approved
commercial extract of M. pachydermatis in healthy
dogs without an overgrowth of Malassezia and in atopic
dogs with and without an overgrowth of M. pachydermatis
via IDT and an ELISA technique, and to determine
the optimal (threshold) concentration of the allergenic
extract for IDT.

MATERIALS AND ME T HODS


Animals
All experiments were approved by the Institutional
Animal Care and Use Committee at the University of
Pennsylvania.
Twelve privately owned, clinically normal dogs were
tested for reactivity to a commercial M. pachydermatis
extract (see below for specifics), and serum was collected for an anti-M. pachydermatis IgE ELISA assay.
Inclusion criteria for normal subjects included the following: normal dermatological examination with no
history of pruritus, skin or ear disease, and lack of
cytological evidence of excessive Malassezia colonization of the skin and ear canals. For these purposes,
excessive colonization of the skin was defined as 10
Malassezia organisms per 1.6 cm2 area on a microscopic

glass slide containing an acetate tape preparation of


the skin. Samples were obtained from four anatomical
locations: the interdigital space of a forepaw, the ventral area of the neck, an axillary fossa, and the inguinal
region.5 The acetate tape preparation was performed
by gently clipping the area with a no. 40 blade and
gentle removal of the hair with a dry gauze sponge. The
tape was then pressed firmly against the skin two times
in succession, labelled, and stained by modified Wrights
stain (Diff-Quik, Dade AG, Ddingen, Switzerland),
for subsequent microscope evaluation.13 Ear cytology
was obtained by a cotton swab of each external ear
canal, at the transition between the vertical and horizontal canal. The swab specimens were then rolled on to
a glass microscope slide, labelled, heat fixed and stained
by modified Wrights stain (Diff-Quik, Dade AG), for
subsequent microscope evaluation. Excessive colonization of the ear canals was defined as 5 yeast organism
per 100 oil immersion field of exudate from each ear
canal.14,15 Dogs were also excluded if they had received
steroidal medications orally, parenterally, or topically
within the last 30 days, injectable steroids within
3 months, antihistamines within 14 days, or if they had
been bathed within the last 14 days. The 12 normal
dogs included eight spayed females and four castrated
males. Their ages ranged from 1 to 12 years (median
age 5 years). Normal dogs were not intradermally tested
for the standard panel of aeroallergens.
For the study group, 31 client-owned dogs were
recruited from the small animal dermatology clinic at
the Matthew J. Ryan Veterinary Hospital of the University of Pennsylvania. Inclusion criteria for the study
group were a clinical diagnosis of AD based upon compatible history and clinical signs, and exclusion of other
causes of pruritus.11,12 Coat brushings, skin scrapings
and miticide trial therapies were used to rule out ectoparasitism on all dogs. Dogs with nonseasonal pruritus
underwent dietary elimination trials of 8 weeks minimum duration on either home-prepared or commercial
hydrolysed dog foods to rule out adverse food reaction.
Withdrawal from medications and bathing was as
described for normal dogs (see above). Dogs suspected
of AD were tested intradermally with a standard panel
of 62 commercial aero- and insect allergens. Dogs were
sedated with 750 mg m21 medetomidine (Dormitor;
Pfizer Animal Health, New York, NY, USA), and hair
was clipped from the lateral hemithorax. Histamine
1 : 100 000 w/v (positive control), phenol-buffered saline
(negative control) and each allergen were injected intradermally at a volume of 0.05 mL. Test sites were evaluated
for a wheal and flare response subjectively on the basis
of erythema, induration, turgidity, and surface area at
15 and 30 min postinjection. Reactions were graded on
a scale of 04+ (0 = no reaction compared to the
saline control; 4+ = the maximum reaction compared
to a histamine phosphate control). Wheal and flare
reactions 2+ were considered clinically relevant.
Only the dogs with at least three positive reactions to
the commercial aeroallergens on the intradermal skin
test were included in the atopic group.

2005 European Society of Veterinary Dermatology, Veterinary Dermatology, 16, 261268

Malassezia hypersensitivity

Allergen

M. pachydermatis (Strain 92643, established from a


clinical isolate obtained from the ear canal of an atopic
dog) was grown in liquid trypticase medium (Greer laboratories, Lenoir, NC, USA) for 17 days at 2530 C.
The identity of the isolate was based on the following
microscopic and biochemical profile: globose to ellipsoidal yeast-like conidia that are round at one end and
blunt at the other. From the blunt end, bottle-shaped,
unipolar budding occurs from the phialides with small
collarettes. Biochemical tests (API20C, bioMerieux
Vitek, Hazelwood, MO, USA) showed an assimilation
reaction profile consistent with Malassezia sp. Fatty
acids were not required for growth.
The culture filtrate was separated from the cells by
filtration, concentrated and dialysed by ultrafiltration
using an Amicon H10P5 hollow fibre and lyophilized.
The cellular fraction, recovered by filtration was further
processed by washing in acetone and Ball-milled to
prepare a powder for extraction. The acetone-powdered
cells were extracted at 1 : 10 w/v in 0.1 M ammonium
bicarbonate pH 7.8 for 18 h at 28 C. The extract was
centrifuged at 10 000 g for 30 min, and the supernatant
recovered and dialysed for 48 h in a 6000 MW cut-off
Spectra/Por dialysis membrane (Spectrum Laboratories, Rancho Dominguez, CA, USA) against 0.01 M
ammonium bicarbonate pH 7.8 at 28 C. The resulting extract was clarified by filtration and lyophilized.
Extracts used for skin testing were prepared by extracting the acetone cell powder at 1 : 10 w/v in Cocas
extraction fluid (5 g L1 NaCl; 5.4 g L1 NaHCO3;
0.4% v/v phenol) for 18 h at 28 C. The extract was
clarified by centrifugation and sterilized by filtration
through a 0.2 m Millipore membrane filter. Sterility
and safety testing was performed as part of lot release
testing and in conformance with Greers USDA license
for allergen extract manufacturer.
The Malassezia pachydermatis extract was diluted
with phosphate-buffered saline to obtain the following
seven test concentrations: 4000 PNU (protein nitrogen
units) mL1, 2000 PNU mL1, 1000 PNU mL1,
500 PNU mL1, 250 PNU mL1, 100 PNU mL1 and
50 PNU mL1 for intradermal testing. The protein
nitrogen unit (PNU) of the extracts was determined
using the Kjeldahl method after precipitation of protein using phosphotungstic acid. The diluted allergens
were stored in glass vials and new dilutions were made
every 2 weeks.

Intradermal testing
For both normal and atopic dogs, 0.05 mL of each of the
dilutions of allergen (4000 PNU mL1, 2000 PNU mL1,
1000 PNU mL1, 500 PNU mL1, 250 PNU mL1,
100 PNU mL1 and 50 PNU mL1) was injected, along
with the positive control (1 : 100 000 w/v histamine),
and the negative control (phenol-buffered saline). The
31 dogs with AD were tested immediately after their
standard IDT. Test sites were evaluated subjectively by
the same investigator for a wheal and flare response
and based upon erythema, induration, turgidity, and

263

surface area after 15 and 30 min postinjection. Evaluation of reactions utilized the same scale as for the
standard panel of aeroallergens.

Cytology
All atopic dogs were evaluated for cytological evidence
of M. pachydermatis after IDT evaluation was completed. Acetate tape preparations were collected by the
standard technique from at least four of the following
anatomical locations: the most erythematous interdigital space, ventral neck fold, axillary fossa, inguinal
region, periocular, perineal, and perioral regions.5,13
Samples from both external ear canals were transferred
to glass microscope slides by swab and heat fixed. The
samples were stained using the modified Wrights stain
(Diff-Quik, Dade AG), and examined at 100 objective under oil immersion. Dogs with clinically significant Malassezia overgrowth of the skin (MD) and ears
(MO), respectively, were diagnosed when at least one
slide was positive for yeast as defined by 10 yeast
organisms/1.6 cm2 (MD) or 5 yeast organisms on
average per 100 oil immersion field (MO).14 Absence
of MD and MO were determined by the same cytological criteria used for the clinically normal dogs.
The atopic dogs were then grouped on the basis of
results of the IDT and cytological evaluation into three
groups: atopic dogs with MD (MD+), atopic dogs with
only MO (MDMO+), and atopic dogs with neither
MD nor MO (atopic control dogs) (MDMO).

Sera collection
Five ml of blood was collected by jugular venepuncture
from all dogs and sera were separated by centrifugation
and stored at 80 C until used for ELISA assay.

Enzyme-linked immunosorbant assay


The enzyme-linked immunosorbent assay (ELISA)
used throughout this study is a noncompetitive, solidphase enzyme immunoassay that incorporates a
mixture of biotinylated monoclonal anticanine IgE
antibodies as the primary tracer for allergen-specific
IgE molecules. Polystyrene microtitre wells (Immulon
4HBX, Thermo Laboratory systems, Franklin, MA,
USA) were coated overnight at 4 C with either the
Malassezia cellular, culture filtrate, or the Greer skin
test preparation, each at a concentration previously
shown in preliminary experiments to saturate the wells.
These determined extract concentrations were as
follows: 1 : 500 dilution of 1 mg mL1 cellular extract
preparations, 1 : 300 dilution of 3 mg mL1 culture filtrate
preparation, and 1 : 100 dilution of 20 000 PNU mL1
of the Greer skin test preparation.
Preliminary data not shown here verified the following ELISA conditions: ELISA plate selection (Immulon 4 was chosen out of the following compared
plastics: 1B, 2B, 4B Immulon and NUNO0), the coating condition with each extract, the anti IgE dilution
factor, incubation time, and the sera dilution factor.
Plate wells without allergen served as background
controls; positive and negative control serum directed

2005 European Society of Veterinary Dermatology, Veterinary Dermatology, 16, 261268

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K Farver et al.

towards June grass pollen allergens served as calibrators to normalize the ELISA results.
No true positive reference sample exists for Malassezia antigen ELISA. But a pooled sera was used to
simulate a positive reference sample. This reference
sample included sera from five of the atopic cases with
Malassezia-associated dermatitis (MD+) who reacted
with a positive wheal and flare response of 2 to all
dilution concentrations tested (4000 PNU mL1, 2000
PNU mL1, 1000 PNU mL1, 500 PNU mL1, 250
PNU mL1, 100 PNU mL1, and 50 PNU mL1).
Unreacted sites were blocked with a 1% solution of
neutral monoethanolamine. For evaluation, 100 L of
3-fold serial dilutions of each serum sample in 50 mM
TRIS buffered saline pH 7.5 was added to duplicate
wells. Following an overnight incubation at 4 C in a
humidified chamber, the wells were washed for four
cycles with TRIS-buffered saline containing 0.05%
Tween 20. Biotinylated anti-IgE (Greer Laboratories,
Lenoir NC) (100 L), diluted in TRIS buffered saline
pH 7.5, was added to each well and incubation was
continued for 2 h at room temperature (22 C). Unreacted anti-IgE-biotin was removed by washing and
100 L of streptavidin alkaline phosphatase was added
to each well. Following a 1-h incubation period the
wells were washed (4) as described above, and 100 L
of p-ntirophenyl phosphate (pNPP) substrate was
added to each well. Substrate development was
allowed to continue for precisely 1 h. The reaction
was stopped by adding 50 L of 20 mM cysteine to each
well. The absorbance of each well was measured at
405 nm using a Molecular Device (Sunnyvale, CA,
USA) Versamax tunable microplate reader. The
absorbance of each well on the plate was normalized
on the basis of the calibrators. Each individual serum
background control value was then subtracted from
the results for each sample. Finally, the two duplicate
wells were averaged. This net result was the reported
mean adjusted absorbance value.

Statistical analysis
To determine differences between the atopic dogs in the
group without MD (MD) and with MD (MD+) for
intradermal wheal and flare response 2 at each dilution, the Fishers exact test was used. To determine differences between MD and MD+ for the single sera
ELISA results, the Students t-test was used. All analyses were performed using SAS statistical software
(Version 9.1, SAS Institute, Cary, NC, USA). A Pvalue of 0.05 was considered statistically significant.
There were not enough animals in the other two groups
to make a statistical comparison. Only MD and MD+
groups were compared.

R E SU LTS
All dogs in all groups reacted to the positive control
with a significant reaction. None of the dogs in any of
the groups reacted significantly to the negative control.

Figure 2. Percentage of atopic and normal dogs in each of the four


groups with a positive reaction of 2 on IDT at each Malassezia
dilution concentration.

In the group of 12 healthy nonatopic dogs, at the concentration of 4000 PNU mL1 four dogs reacted with
scores 2. At the concentration of 2000 PNU mL1 three
of the dogs reacted with scores 2. None of the dogs
reacted at the lower concentrations of 1000 PNU mL1,
500 PNU mL1, 250 PNU mL1, 100 PNU mL1, and
50 PNU mL1. Therefore, the optimal (threshold)
concentration was 1000 PNU mL1. This is because it is
the highest concentration where greater than 90% of
normal dogs failed to react. This was chosen as the concentration for use in evaluating differences between
atopic dogs with and without MD.
Cytology results divided the 34 atopic dogs into the
following groups. There were 15 dogs that had an overgrowth of Malassezia on their skin (MD+). Four of the
15 dogs in this MD+ group also had an overgrowth of
Malassezia in their external canal. There were 16 dogs
that did not have an overgrowth of yeast on their skin
or in their ears. (MDMO). Finally, there were three
dogs that did not have MD but had MO (MDMO+).
This atopic group (MDMO+) was too small to be
compared statistically to the other two groups.
Intradermal test results demonstrated that in the
MDMO+ group, all three of the dogs reacted at the
calculated threshold dilution of 1000 PNU mL1. Of
the 15 atopic dogs in the MD+ group, 14 (93%) reacted
at the threshold dilution of 1000 PNU mL1 whereas
only five of the 16 atopic dogs without MD (MDMO)
(31%) reacted. This was a significant difference
(P < 0.001, Fishers exact test). The dogs in the MD+
group also had significantly stronger wheal and flare
reactions than the MDMO group at less concentrated dilutions (Fig. 2). Of the 4/15 dogs in the MD+
group that also had an overgrowth of Malassezia in the
ears (subgroup MD+MO+), all four of these dogs reacted
with 2 response to the threshold concentration.

2005 European Society of Veterinary Dermatology, Veterinary Dermatology, 16, 261268

Malassezia hypersensitivity

265

Table 1. Mean adjusted absorbance values, as detected by ELISA, for anti-Malassezia IgE in canine sera
ELISA variables

Group

Median

Mean

SD

Max. value

Min. value

# of cases

Culture filtrate sera diluted 1 : 10

MD+ atopics
MDMO atopics
Normal

106
93
139

166
166
149

155
287
60

644
1270
258

31
31
66

15
16
12

Culture filtrate sera diluted 1 : 30

MD+ atopics
MDMO atopics
Normal

56
64
65

121
119
73

180
208
34

734
914
133

0
16
23

15
16
12

Cellular extract sera diluted 1 : 10

MD+ atopics
MDMO atopics
Normal

121
82
109

208
183
112

208
328
84

831
1418
312

0
0
15

15
16
12

Cellular extract sera diluted 1 : 30

MD+ atopics
MDMO atopics
Normal

60
55
55

144
132
73

205
224
42

795
960
145

0
0
30

15
16
12

Greer extract sera diluted 1 : 10

MD+ atopics
MDMO atopics
Normal

167
98
132

206
178
152

164
234
80

706
1039
296

38
38
58

15
16
12

Greer extract sera diluted 1 : 30

MD+ atopics
MDMO atopics
Normal

86
60
61

148
128
72

188
220
43

754
954
151

0
12
18

15
16
12

Upon further historical evaluation of the five cases


in the MDMO group that reacted with a positive
response to the threshold concentration (31%), it was
determined that one had a known episode of MD
7 months prior to admission in the study, while another
one of these had an episode of MO 4 months prior to
study admission.
The ELISA absorbance values were determined for
sera of all dogs. No significant differences were detected
between the groups, at either serum dilution or when
using any of the three different allergen extracts for
antibody capture (Table 1).

D ISCU SSION
The irritant threshold is the highest concentration of
an allergen that does not produce a positive result in
> 90% of normal individuals.11 None of the normal
dogs in this study reacted at 1000 PNU mL1. However,
higher concentrations induced a mean positive wheal
and flare reaction of 2 in more than 10% of the normal dogs, confirming 1000 PNU mL1 to be the irritant
threshold. Previous studies have evaluated the irritant
threshold concentrations of crude extracts of M. pachydermatis. One study found it to be 2 g mL1 for a
lyophilized M. pachydermatis extract and most of its
fractions,8 while another found it to be 2000 g mL1.9 This
illustrates the need for a standardized commercial allergen, as these studies were performed by the same investigator.
The commercial extract used in this study uses the
PNU mL1 assay to standardize the concentration
batch to batch. This assay has been adopted by the
FDA and USDA for labelling of allergenic extracts.
The assay has a repeatability coefficient of variation of
5% and a reproducibility coefficient of variation of
circa 14%. Therefore, it has reasonable repeatability.
When evaluating an extract for its utility in true
allergy detection, the irritant threshold (evaluated in

normal dogs) is used as a reference point for evaluating


allergic dogs. In this study, 93% of the MD+ atopic
dogs and 31% of MDMO atopic dogs were reactive
at the irritant threshold, which demonstrates that the
irritant and allergy detection thresholds are similar
or the same.
There was a strong statistical difference when comparing the two atopic groups. The MD+ dogs had greater
IDT responses than did MDMO dogs (P = 0.00063).
There was also a significant difference between these
two groups at several other dilutions. The proposed
mechanism by which atopic dogs sensitize to Malassezia yeast is epicutaneous contact with allergen
produced by the organism, which induces the atopic
cascade. A likely contributing factor to sensitization by
atopic dogs is impairment of stratum corneum barrier
function due to inflammation and self-trauma. Dogs
with increased numbers of yeast on the skin would be
expected to have a greater chance of sensitization.
However, 31% of the MD group did react on IDT.
There are several possible explanations for this finding
(as reported in a previous study).8
The use of a culture technique has been shown to be
more sensitive than cytological tape staining in identifying the presence of Malassezia on the skin.15 Therefore, if this test had been employed instead of the
cytological tape analysis, it is possible these MD but
IDT+ dogs would have been assigned to the MD+
group, while the rest of the MD group would still have
been assigned to the appropriate group. However, it is
known that Malassezia is a commensal organism on
normal skin, and it is therefore possible that this technique would have raised the amount of false positives
assigned to the MD+ group. Also, to the authors
knowledge, no studies have compared type I hypersensitivity reactions between atopics with and without
culture positive Malassezia, whereas cytological staining
has been shown to consistently find significant differences between these two groups.79

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K Farver et al.

These dogs may have been sensitized by a previous


episode of MD or MO that subsequently cleared
before enrolment in the study. One of the five cases in
the MDMO group, which reacted with a positive
response to the threshold concentration, had a known
episode of MD 7 months prior to admission in the
study, while another case in this group had an episode
of MO 4 months prior to study admission. Whether
MO contributes to type I hypersensitivity in atopic
dogs is as yet unknown. Malassezia yeast is commonly
associated with otitis externa in atopic dogs.14 All of
the dogs in the MDMO+ group in this study reacted
with a positive reaction to the irritant threshold. This
is in contrast to previous findings, where two dogs in an
MDMO+ group did not react upon IDT.7 In the previous study, it was postulated that the dogs in the MD
MO+ group did not elicit a type I hypersensitivity reaction because of the possible protective microenvironment of the ear. They postulated that high wax and
lipid content in the ear perhaps interfered with recognition of the organism by the local T-cell population.
The results of this study do not support this theory.
However, the case numbers of this particular group in
both studies are too low for statistical analysis. Elucidation of the role of Malassezia hypersensitivity in
otitis will require further study.
In addition, this studys cytological definition of
Malassezia overgrowth may not be sufficiently sensitive in identifying the number of organisms required
for sensitization. The number of organisms used to define
MD was based upon cytological analysis of healthy dogs
with normal skin and may not account for the physiological and microenvironmental changes that occur in
inflamed skin that could allow fewer organisms to exert
a pathogenic role. It may be this group for which positive IDT is most valuable, as these dogs may be sensitized to Malassezia yeast but negative on cytology by
our current definition. Future studies evaluating the
response to antifungal therapy of MDIDT+ dogs are
indicated.
In this study, IDT showed a strong discrimination
between groups whereas ELISA did not detect a difference between groups. Immediate IDT reactivity and
antigen specific IgE serology both attempt to measure
humoral responses to allergens. Therefore, one would
expect findings to be at least correlative. However, it is
not uncommon to have conflicting results for environmental aeroallergens between IDT and in vitro test
results.16,17 There are several factors that could have
contributed to the lack of correlation between the two
tests studied here.
Concentrations of anti-Malassezia IgE were distributed similarly in all groups. The relatively high
amounts of circulating antibody in the MD atopics
and normal dogs may have been a result of prior
episodes of MD or MO that were unknown in the history
and inactive at the time of study enrolment. Similarly,
the relatively high/moderate IgE levels documented in
the normal group may reflect a normal response seen in
healthy dogs to commensal populations.18 However,

this contrasts with the results of another study which


found significantly higher IgE levels in the sera of
atopic dogs than in normal dogs.6
The differences between the two tests studied here
could also be due to the heterogenicity of IgE, which
has been documented in dogs. Two subforms of canine
IgE have been identified. One of these subforms has
been shown to be less reactive to monoclonal anticanine IgE antibody in ELISA in comparison to a polyclonal anti-IgE antibody.19 It is possible that the
monoclonal antibody we used did not recognize the
same Malassezia-specific IgE that was responsible for
in vivo responsiveness on IDT.
The lack of correlation between the two tests may
also be due to the ELISA design and further modification may be needed before it can be offered for reliable
screening of atopic patients with MD. One potential
problem may be the lack of specificity caused by using
crude extracts for preparing the allergosorbents. Use of
selected purified Malassezia allergens could increase
the specificity of the assay.
In conclusion, this study confirms that many atopic
dogs develop type I hypersensitivity to M. pachydermatis,
and that intradermal testing with Greer Laboratories
M. pachydermatis extract may be useful in clinical evaluation of these patients. Future studies should focus on
enhancing the ELISAs capabilities and in identifying
the major allergens within this extract that are recognized by atopic dogs with Malassezia hypersensitivity.
Studies of yeast cross-reactivity with environmental
fungi should also be considered.

AC K N OW L E D G E M E N T
Greer Laboratories, Lenoir, NC, USA provided the
Malassezia allergen for IDT, and the laboratory equipment for the ELISA evaluation.

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Rsum Malassezia pachydermatis est considre comme un facteur contribuant aux lsions de dermatite
atopique canine (AD). Le but de cet essai tait dtudier la rponse humomrale un extrait commercial de
M. pachydermatis. Quinze chiens atopiques avec une prolifration de Malassezia sur la peau (MD), 16 chiens
atopiques sans MD, trois chiens atopiques avec prolifration de Malassezia dans les oreilles seulement (MO), et
12 chiens normaux ont t tests par voie intradermique avec un extrait de M. pachydermatis (Greer laboratories,
Lenoir, NC) 50, 100, 250, 500, 1000, 2000, et 4000 PNU/ml. Tous les chiens ont t valus cytologiquement
par test la cellophane adhsive et couvillonage auriculaire pour dterminer la prsence de MD ou MO. Un
dosage des IgE anti-Malassezia a t ralis dans tous les cas en utiliasnt trois extraits de Malassezia par test
ELISA. La concentration irritante des extraits laquelle tous les chiens sains non atopiques ne ragissaient plus
tait de 1000 PNU/ml. Une diffrence significative de ractivit intradermique a t observe entre les groupes
de chiens atopiques. A cetet dilution, 93% (14/15) des chiens atopiques du groupe MD, 31% (5/16) des chiens
du groupe atopique sans MD ou MO, et 100% (3/3) des chiens atopiques MO ont ragi. Aucune diffrence
significative pour les taux dIgE sriques par ELISA na t observe. Ces rsultats suggrent que lextrait de
M. pachydermatis commercialis par Greer est utile pour tester par voie intradermique les chiens phnotype
allergique, et que les chiens atopiques MD sont plus risque davoir une raction dhypersensibilit de type I
Malassezia que les chiens sans MD. Le test ELISA en revanche ncessite des tudes supplmentaires pour tre
utile dans le diagnostic des hypersensibilits Malassezia.
Resumen Se considera que Malassezia pachydermatis es un factor que contribuye a producir dermatitis atpica
(AD). El objetivo de este estudio fue investigar la respuesta humoral a extracto comercial de M. pachydermatis.
Quince perros atpicos con sobrecrecimiento de Malassezia en la piel (MD), 16 perros atpicos sin MD, tres perros
atpicos con sobrecrecimiento de Malassezia slo en los odos (MO), y 12 perros normales fueron probados va
intradrmica con extracto de M. Pachydermatis (Laboratorios Greer, Lenoir, North Carolina, USA) a concentraciones de 50, 100, 250, 500, 1000, 2000 y 4000 PNU/ml. Todos los perros se evaluaron mediante citologa de
piel obtenida con cinta adhesiva y mediante muestreo bilateral del exudado de los odos para determinar la presencia de MD o MO. Se evalu el suero de los perros para la presencia de IgE frente a Malassezia utilizando tres
extractos de Malassezia en un ensayo de ELISA. El nivel de irritante al cual los perros sanos no atpicos dejaron
de reaccionar fue 1000 PNU/ml. Se encontr una diferencia significativa en la reaccin intradermal entre los
diferentes grupos de perros atpicos. A esta dilucin, 93% (14/15) de los perros atpicos con MD, 31% (5/16) de
los perros atpicos sin MD ni MO, y 100% (3/3) de los perros atpicos slo con MO reaccionaron al extracto.
No observamos diferencias significativas en los niveles de IgE en el suero entre los distintos grupos, valorados
mediante el ensayo de ELISA de laboratorios Greer utilizando cualquiera de los tres extractos. Estos resultados
indican que el extracto de M. Pachydermatis de los laboratorios Greer es til para la prueba intradrmica de los
perros con fenotipo alrgico, y que es ms probable que los perros alrgicos con MD presenten hipersensibilidad
2005 European Society of Veterinary Dermatology, Veterinary Dermatology, 16, 261268

268

K Farver et al.
de tipo I frente a Malassezia que los perros sin MD. El ensayo de ELISA posiblemente requiera una mejor
caracterizacin para ser til en el diagnstico de hipersensibilidad frente a Malassezia.
Zusammenfassung Malassezia pachydermatis wird als beitragender Faktor zur caninen atopischen Dermatitis
betrachtet. Das Ziel dieser Studie war die Untersuchung der humoralen Antwort auf einen kommerziell produzierten M. pachydermatis Extrakt. Fnfzehn Hunde mit bermssigem Wachstum von Malassezien auf der Haut
(MD), 16 atopische Hunde ohne MD, 3 atopische Hunde mit bermssigem Wachstum von Malassezien nur in
den Ohren (MO), und 12 normale Hunde wurden mit M. pachydermatis Extrakt (Greer Laboratories, Lenoir,
NC) mit Konzentrationen von 50, 100, 250, 500, 1000, 2000, und 4000 PNU/ml intradermal getestet. Alle Hunde
wurden auch zytologisch untersucht mittels Klebestreifenabklatsch und Probenahme des Exsudates aus beiden
Ohren, um das Vorhandensein von MD oder MO zu ermitteln. Bei jedem Hund wurde Serum auf anti-Malassezia
IgE untersucht mittels dreier Malassezien Extrakte in einem ELISA Test. Die reizende Grenzwertkonzentration,
bei der gesunde nicht atopische Hunde aufhrten zu reagieren, lag bei 1000 PNU/ml. Es bestand ein signifikanter
Unterschied bei den Reaktionen im Intradermaltest zwischen den atopischen Gruppen. Bei dieser Verdnnung
reagierten 93% (14/15) der atopischen MD Gruppe, 31% (5/16) der atopischen Gruppe ohne MD oder MO, und
100% (3/3) der atopischen MO Gruppe. Zwischen den verschiedenen Gruppen bestand kein signifikanter Unterschied im Bezug auf Serum IgE Werte, die mittels Greer ELISA Test unter Verwendung der drei unterschiedlichen
Extrakte gemessen wurden. Diese Ergebnisse besttigen, dass Greers M. pachydermatis Extrakt ntzlich ist fr
das intradermale Testen von Hunden mit einem allergischen Phnotyp und dass Atopiker mit MD hufiger eine
Malassezien Hypersensibilitt vom Typ-1 aufweisen als solche ohne. Um fr die Diagnose einer Malassezien
berempfindlichkeit ntzlich zu sein, bedarf es mglicherweise einer Weiterentwicklung des ELISA Tests.

2005 European Society of Veterinary Dermatology, Veterinary Dermatology, 16, 261268